A Split-Abl Kinase for Direct Activation in Cells.
A Split-Abl Kinase for Direct Activation in Cells.
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DOI：
10.1016/j.chembiol.2017.08.007
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发表时间：
2017-10-19
影响因子：
8.6
通讯作者：
Wells JA
中科院分区：
文献类型：
--
作者：
Diaz JE;Morgan CW;Minogue CE;Hebert AS;Coon JJ;Wells JA
为了剖析单个激酶的细胞作用，设计用于它们选择性激活的工具是有用的。我们描述了分裂cAbl激酶（sKin-Abl）的工程设计，该激酶在细胞中用雷帕霉素快速激活，并允许时间、剂量和区室化控制。我们的设计策略涉及在哺乳动物细胞中的经验筛选和在N叶中的分裂位点的鉴定。该分裂位点导致活性完全丧失，其可以在细胞中小分子诱导的二聚化后恢复。值得注意的是，分裂位点可转运到相关的Src Tyr激酶和远亲的Ser/Thr激酶，AKT，这表明激酶有更广泛的应用。为了定量磷酸酪氨酸（pTyr）修饰的诱导倍数，我们采用了定量蛋白质组学，NeuCode-SILAC。我们鉴定了许多已知的Abl底物，包括自磷酸化位点和新的pTyr靶点，总共432个pTyr位点。我们相信这种分裂激酶技术将有助于直接激活细胞中的蛋白激酶。迪亚兹，J.，和摩根，C.，等人详细描述了他们将cAbl激酶工程化为小分子可活化变体，该变体在哺乳动物细胞中被快速和选择性地活化，并继续利用NeuCodeSILAC定量磷酸蛋白质组学来鉴定已知底物并暴露cAbl活化的已知和新的下游靶标。
To dissect the cellular roles of individual kinases, it is useful to design tools for their selective activation. We describe the engineering of a split-cAbl kinase(sKin-Abl) that is rapidly activated in cells with rapamycin and allows temporal, dose, and compartmentalization control. Our design strategy involves an empirical screen in mammalian cells and identification of split site in the N-lobe. This split site leads to complete loss of activity, that can be restored upon small molecule-induced dimerization in cells. Remarkably, the split site is transportable to the related Src Tyr kinase and the distantly related Ser/Thr kinase, AKT, suggesting broader applications to kinases. To quantify the fold-induction of phosphotyrosine(pTyr) modification, we employed quantitative proteomics, NeuCode-SILAC. We identified a number of known Abl substrates, including autophosphorylation sites and novel pTyr targets, 432 pTyr sites in total. We believe this split-kinase technology will be useful for direct activation of protein kinases in cells. Diaz, J., and Morgan, C., et al. detail their engineering cABL kinase into a small molecule activatable variant that is rapidly and selectively activated in mammalian cells and proceed to utilize NeuCode SILAC quantitative phosphoproteomics to identify known substrates and expose known and novel downstream targets of cAbl activation.
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影响因子：
7.3
作者：
Colicelli J
通讯作者：
Colicelli J
影响因子：
48
作者：
Hebert, Alexander S.;Merrill, Anna E.;Bailey, Derek J.;Still, Amelia J.;Westphall, Michael S.;Strieter, Eric R.;Pagliarini, David J.;Coon, Joshua J.
通讯作者：
Coon, Joshua J.
影响因子：
8.8
作者：
Baughman JM;Rose CM;Kolumam G;Webster JD;Wilkerson EM;Merrill AE;Rhoads TW;Noubade R;Katavolos P;Lesch J;Stapleton DS;Rabaglia ME;Schueler KL;Asuncion R;Domeyer M;Zavala-Solorio J;Reich M;DeVoss J;Keller MP;Attie AD;Hebert AS;Westphall MS;Coon JJ;Kirkpatrick DS;Dey A
通讯作者：
Dey A
DOI：
10.1126/science.aah3404
发表时间：
2016-12-16
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Dagliyan O;Tarnawski M;Chu PH;Shirvanyants D;Schlichting I;Dokholyan NV;Hahn KM
通讯作者：
Hahn KM
影响因子：
46.9
作者：
通讯作者：
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